U.S. patent application number 12/362375 was filed with the patent office on 2010-07-29 for microfluidic glycan analysis.
Invention is credited to Magdalena A. Bynum, Rudolf Grimm, Kevin P. Killeen, Karia M. Robotti.
Application Number | 20100190146 12/362375 |
Document ID | / |
Family ID | 42317619 |
Filed Date | 2010-07-29 |
United States Patent
Application |
20100190146 |
Kind Code |
A1 |
Bynum; Magdalena A. ; et
al. |
July 29, 2010 |
Microfluidic Glycan Analysis
Abstract
Microfluidic devices and methods for analyzing glycan profiles
of glycoproteins are provided. Some embodiments of the devices
comprise a deglycosylation column for cleaving glycans, an optional
cleaning column for removing proteins, a trapping column for
enriching glycans, and a separation column for resolving glycans.
The devices and methods significantly improve the speed and
sensitivity of glycan analysis.
Inventors: |
Bynum; Magdalena A.;
(Mountain View, CA) ; Grimm; Rudolf; (San Jose,
CA) ; Killeen; Kevin P.; (Woodside, CA) ;
Robotti; Karia M.; (Cupertino, CA) |
Correspondence
Address: |
AGILENT TECHNOLOGIES INC.
INTELLECTUAL PROPERTY ADMINISTRATION,LEGAL DEPT., MS BLDG. E P.O.
BOX 7599
LOVELAND
CO
80537
US
|
Family ID: |
42317619 |
Appl. No.: |
12/362375 |
Filed: |
January 29, 2009 |
Current U.S.
Class: |
435/4 ; 204/451;
204/601; 435/288.6; 435/299.1 |
Current CPC
Class: |
B01L 2200/10 20130101;
G01N 27/44791 20130101; G01N 30/7266 20130101; H01J 49/00 20130101;
B01L 3/502753 20130101; B01L 2400/0487 20130101; G01N 30/465
20130101; B01L 3/502738 20130101; G01N 2030/085 20130101; B01L
2300/0887 20130101; B01L 2400/0644 20130101; F16K 99/0001 20130101;
G01N 30/6095 20130101; B01L 2300/0874 20130101; B01D 15/08
20130101; B01L 2300/0627 20130101; B01L 2400/0421 20130101; G01N
1/405 20130101; G01N 2400/00 20130101; G01N 33/6842 20130101; B01L
3/502761 20130101; B01L 2400/0406 20130101 |
Class at
Publication: |
435/4 ;
435/299.1; 435/288.6; 204/601; 204/451 |
International
Class: |
G01N 27/26 20060101
G01N027/26; C12M 3/04 20060101 C12M003/04; C12M 1/34 20060101
C12M001/34; C12Q 1/00 20060101 C12Q001/00 |
Claims
1. A microfluidic device for removing carbohydrate from a
glycoprotein, comprising: a deglycosylation column comprising a
solid support and an enzyme immobilized to the solid support,
wherein the enzyme is capable of cleaving carbohydrates from
glycoproteins; a trapping column that is capable of binding
carbohydrates; a separation column capable of separating
carbohydrates; and a plurality of inlet/outlet ports; wherein said
ports are configured so that when said device is coupled with a
switching element that comprises at least one channel, the
combination of said ports, columns and at least one channel forms a
valve system that can be switched between at least a first state
and a second state, the first state allowing fluid communication
between the deglycosylation column and the trapping column, and the
second state allowing fluid communication between the trapping
column and the separation column.
2. The device of claim 1, further comprising a cleaning column
capable of binding proteins, wherein the cleaning column is
configured to be connectable to the deglycosylation column and/or
the trapping column by the valve system.
3. The device of claim 1, wherein the enzyme is N-glycosidase
F.
4. The device of claim 1, wherein the solid support in the
deglycosylation column comprises beads or a monolithic medium.
5. The device of claim 1, wherein the separation column is a liquid
chromatography column.
6. The device of claim 1, wherein the separation column is a
capillary electrophoresis apparatus.
7. The device of claim 1 that comprises two layers, wherein the
deglycosylation column is in one layer, and the trapping column and
separation column are in the other layer.
8. The device of claim 2 that comprises three layers, wherein the
deglycosylation column is in a first layer, the cleaning column is
in a second layer, and the trapping column and separation column
are in a third layer.
9. A system for analyzing a sample, comprising the device of claim
1, the switching element, and a mass spectrometer.
10. The system of claim 9, wherein the mass spectrometer comprises
an electrospray ion source.
11. A method for analyzing the carbohydrate moieties of
glycoproteins, comprising: applying a sample that may comprise
glycoproteins to the device of claim 1; digesting the glycoproteins
in the deglycosylation column to result in cleaved carbohydrates;
binding the cleaved carbohydrates to the trapping column; eluting
the cleaved carbohydrates from the trapping column; and separating
the cleaved carbohydrates with the separation column.
12. The method of claim 11, further comprising removing proteins
after the digesting with a cleaning column capable of binding
proteins.
13. The method of claim 11, wherein the cleaved carbohydrates are
separated by liquid chromatography.
14. The method of claim 11, wherein the cleaved carbohydrates are
separated by capillary electrophoresis.
15. The method of claim 11, further comprising analyzing the
cleaved carbohydrates using mass spectrometry.
16. The method of claim 11, wherein the sample contains up to 50 ng
of glycoproteins.
17. The method of claim 11, wherein the method is completed within
10 minutes.
18. The method of claim 11 that is performed under conditions that
allow at least some of the cleaved carbohydrates to remain in amino
glycan forms.
19. The method of claim 11, wherein the glycoproteins are digested
with N-glycosidase F.
20. A kit for glycan analysis, comprising the device of claim 1 and
at least one reagent for sample dilution or column elution.
Description
BACKGROUND OF THE INVENTION
[0001] The analysis of glycoprotein structure and function, known
as glycomics, has become a new and important area of research.
Glycosylation is the most common post-translational modification of
cell surface and extracellular matrix proteins. Glycoproteins play
an important role in cell-adhesion and immune response. Changes in
abundance and glycan profiles have been correlated with progression
of diseases, such as cancer and rheumatoid arthritis. In addition,
the analysis of glycan profiles is critical in the bio-therapeutic
industry. For example, antibody bio-therapeutics contain
glycosylated amino acids that assist in maintaining drug activity
and preventing drug rejection by the immune system. Therefore,
companies that make these bio-therapeutics have to monitor and
verify their glycan profiles.
[0002] Glycan analysis is usually performed by capillary
electrophoresis or mass spectrometry methods. In either case, the
glycans must be removed from the glycoproteins, separated and
analyzed. The process of removing glycans from glycoproteins
traditionally includes an in-solution enzymatic reaction with
PNGase F that requires a 24-hour incubation time. After the enzyme
reaction, protein precipitation is needed to separate the glycans
from the proteins for analysis of the glycans. Finally, the free
glycans are analyzed with mass spectrometry or capillary
electrophoresis. These steps are time-consuming and cumbersome, and
the manual operations are error-prone. Furthermore, a relatively
large quantity of starting materials is required to generate high
quality data. Therefore, a better way of performing glycan analysis
is desirable.
BRIEF DESCRIPTION OF THE DRAWINGS
[0003] FIG. 1A is a schematic diagram of a top-down view of a
microfluidic chip that can be used for glycan analysis of
glycoproteins. FIG. 1B is a schematic diagram of a top-down view of
a switching element in the form of a rotor that can be coupled with
the chip shown in FIG. 1A. FIG. 1C shows one state of a valve
system formed when the chip of FIG. 1A is coupled with the rotor of
FIG. 1B. In this state, the trapping column is connected to the
deglycosylation column. FIG. 1D shows another state of the valve
system in which the trapping column is connected to the separation
column.
[0004] FIG. 2 shows two layers of a microfluidic chip.
[0005] FIG. 3 shows a three-dimensional view of how deglycosylation
and glycan enrichment occur in a microfluidic device of the present
application. Some channels from a switching element, which is
coupled with the device, are also shown.
[0006] FIG. 4 shows a three-dimensional view of a microfluidic
device switched to a position to send collected glycans to a
separation column. Some channels from a switching element, which is
coupled with the device, are also shown.
[0007] FIGS. 5 and 6 illustrate a three-dimensional view of a
microfluidic device that comprises three layers. Some channels from
a switching element, which is coupled with the device, are also
shown.
[0008] FIG. 7 shows the mass spectral scan of an antibody sample
that has been processed on-chip (right-hand side) or in-solution
(left-hand side).
[0009] FIG. 8 shows the chemical mechanism of enzymatic action of
PNGase F.
[0010] FIG. 9 shows the identity of the glycans corresponding to
the peaks in the mass spectral scan shown in FIG. 7.
[0011] FIG. 10 demonstrates separation of glycan isoforms G1' and
G1'' after a short gradient in liquid chromatography.
DESCRIPTION OF THE INVENTION
[0012] The present invention provides a new, fast, and streamlined
approach to preparing, separating and analyzing glycans using a
microfluidic device. Moreover, due to the design of the
microfluidic device of this invention, a higher recovery yield of
glycans and superior chemical separation of isoforms are achieved,
and a significantly smaller amount of starting material is
required, compared to traditional methods.
[0013] Prior to describing the invention in further detail, the
terms used in this application are defined as follows unless
otherwise indicated.
Definition
[0014] In this specification and the appended claims, the singular
form "a," "an," and "the" include plural reference unless the
context clearly dictates otherwise.
[0015] A "microfluidic device" is a device comprising chambers
and/or channels of micron or submicron dimensions that allow
passage of fluid. The chambers or channels are generally 1 .mu.m to
less than 1000 .mu.m in diameter (or, if not circular, the largest
dimension of the cross section), such as 1 .mu.m to 500 .mu.m, 10
.mu.m to 300 .mu.m, 50 .mu.m to 250 .mu.m, by way of examples.
[0016] A "column" is an apparatus for a particular purpose, usually
for preferentially binding or retaining a substance or class of
substances. Typically, a column comprises a housing in which a
filling is located, and the filling is capable of preferentially
binding or retaining a substance or class of substances. In some
embodiments, the filling may merely provide a medium for various
substances to move through, while the substances move in different
speeds (e.g., due to an electric field). A column (or a housing or
filling) can be of any size, shape or structure, and made of any
material, that is consistent with the purpose of the column.
[0017] As used herein, "connect" may occur by direct or indirect
connections. A direct connection means two objects that are
connected have a physical contact with each other. An indirect
connection means two objects that are connected do not physically
contact each other, but are connected through at least one object
in between.
[0018] As used herein, if two objects are in "fluid communication,"
there is a conduit between the two objects that allows fluid to
flow from one of the objects to the other. A conduit may be a pore,
orifice, opening, channel, tube, or the like.
[0019] The term "carbohydrate" refers to any compound in the
carbohydrate family, including sugars, disaccharides,
oligosaccharides, polysaccharides, simple carbohydrates, complex
carbohydrates, and the like.
Device and Methods
[0020] FIG. 1A illustrates an exemplary device 101 in accordance
with one embodiment of the present invention. The device 101
comprises a deglycosylation column 110 for cleaving glycans from
glycoprotein samples, a trapping column 120 for trapping and
enriching the cleaved glycans, and a separation column 130 for
separating the glycans. Although a particular shape is drawn for
the device 101 and each component of the device, other variations
in shape or relative size can be used for this invention.
Similarly, the other devices and components described in this
disclosure are not limited to the shapes or sizes shown in the
drawings.
[0021] The deglycosylation column 110 comprises a solid support to
which an enzyme is attached, with the enzyme capable of cleaving
carbohydrates from a glycoprotein. In most glycoproteins, the
carbohydrate moiety is attached to the nitrogen of the amide group
in asparagine residues (N-linked glycans), or the oxygen of the
hydroxyl group in serine or threonine residues (O-linked glycans).
Any enzyme that can cleave the carbohydrate moiety from
glycoproteins can be used in the present invention, including
enzymes that are specific for N-linked or O-linked glycans. These
enzymes are known in the art and include, without being limited to,
PNGase F, .beta.-N-Acetyl-glucosaminidase, .alpha.-Fucosidase,
.beta.-Galactosidase, .alpha.-Galactosidase, .alpha.-Neuraminidase,
.alpha.-Mannosidase, .beta.-Glucosidase, .beta.-Xylosidase,
.beta.-Mannosidase, Endo F.sub.1, Endo F.sub.2, Endo F.sub.3, and
Endo H. Materials and methods of immobilizing proteins to solid
supports are also known in the art (see, e.g., Palm and Novotny,
2005). For example, the solid support in the deglycosylation column
110 may be glass or polymer beads, or a monolithic medium (such as
polymethacrylate, polystyrene, polyacrylamide, or the like).
[0022] The trapping column 120 is capable of preferentially binding
carbohydrates but not proteins. For example, hydrophilic
interaction liquid chromatography (HILIC) stationary phase can be
used to retain and desalt the glycans released from glycoproteins
in the deglycosylation column. The separation column 130 is capable
of separating glycans based on their physical and/or chemical
properties. Two main categories of the separation column are liquid
chromatography columns and capillary electrophoresis columns.
[0023] The device 101 also contains a plurality of inlet/outlet
ports 141-146. An inlet/outlet port (or "port") can be a hole,
orifice, opening, any of the above connected to a conduit
(especially a short conduit), or the like, as long as the port
allows fluid to pass from one end of the port to the other. The
ports 141-146 can be used to connect different parts of the device
at different stages when device 101 is aligned with and coupled to
appropriate channels. For example, device 101 can be fit on top of
a rotor 150 that comprises three channels 151, 153 and 155 (FIG.
1B). The rotor 150 can be rotated so that different ports in device
101 are connected by each channel in the rotor when the rotor is in
a different position. For example, the rotor can be rotated to a
position wherein inlet/outlet ports 141 and 142 are connected by
channel 151 (FIG. 1C). At this position, inlet/outlet ports 143 and
144 are also connected (by channel 153), and so are inlet/outlet
ports 145 and 146 (by channel 155). A sample that contains, or is
suspected to contain, glycoproteins is loaded into the
deglycosylation column 110 through an inlet port 111. The
glycoproteins are digested in the deglycosylation column 110,
releasing free glycans as well as the remaining part of the
glycoproteins. This mixture flows to the end of the deglycosylation
column, which is connected to port 141, and enters the trapping
column 120 via channel 151. In the trapping column 120, the glycans
are bound to the carbohydrate-binding substance, while the
remaining components of the mixture flow through the column and are
collected or discarded via channel 155.
[0024] The system is then switched to a different position (see
FIG. 1D), in which the following inlets/outlets are connected: port
142 with port 143, port 144 with port 145, and port 146 with port
141. Thus, the trapping column 120 and the separation column 130
are now in fluid communication through channel 153, and the
deglycosylation column 110 is no longer connected with the trapping
column 120. An elution solution can be added to the trapping column
120 from port 143, and the eluate flows from port 145 to port 144
and enters the separation column 130. The glycans are then
separated in the separation column 130. The outlet of the
separation column 130 can be connected to the sample inlet of a
mass spectrometer to further analyze the glycans by mass
spectrometry.
[0025] Although a rotor is described above as a switching element
to change the fluid communication state of the columns in device
101, other switching elements can be utilized. For instance, a set
of channels and valves can be engaged with device 101 so that, upon
switching one valve or multiple valves, the deglycosylation column
is disconnected from the trapping column, and the trapping column
is connected with the separation column.
[0026] In some embodiments, the deglycosylation column 110 and the
trapping column 120 are continuous. For example, instead of two
discontinuous columns 110 and 120, device 101 can have a chamber
that comprise a deglycosylation material (a deglycosylation enzyme
immobilized to a solid support) in a first part of the chamber to
constitute a deglycosylation column, and a trapping material (which
preferentially binds carbohydrates but not proteins) in the next
part of the chamber to form a trapping column. As another example,
a deglycosylation column can be connected with a trapping column by
an orifice or conduit in device 101, so the two columns are in
fluid communication within the structure of device 101. For these
embodiments, a simpler switching element can be used, which does
not have to connect the deglycosylation column with the trapping
column. It is conceivable that the switching element may comprise a
minimum of a channel and a valve controlling the channel, wherein
the channel can be positioned to connect the trapping column and
the separation column. When the valve is switched off, the trapping
column is not in fluid communication with the separation column. In
this state, sample loading, deglycosylation, and glycan enrichment
(trapping) can be performed. The valve is then switched on to allow
fluid communication between the trapping column and the separation
column for eluting the trapping column and separating the
glycans.
[0027] In some embodiments, different parts of the device are
located on different layers or chips, which are assembled to form
the device (see, e.g., U.S. Patent Publication No. 2006/0171855).
For example, FIG. 2 shows a chip 210 containing the trapping column
120 and the separation column 130. Another chip ("deglycosylation
chip") 220 contains the deglycosylation column. The deglycosylation
chip 220 can be connected to the chip 210 by, for example,
mechanically sealing on top of chip 210, in a manner that allows
fluidic communication between inlet/outlet port 141 of chip 210
with port 141' of deglycosylation chip 220. Note that the
deglycosylation chip 220 does not contain counterparts of
inlet/outlet ports 144 and 145 in this design, because 144 and 145
are used for communication between the trapping column and the
separation column, both of which are on chip 210. Chip 220,
however, may have counterparts of inlet ports 143 and 146 (143' and
146') for alignment purposes only. Thus, port 143 is aligned with
port 143', and port 146 is aligned with port 146' when chips 210
and 220 are assembled.
[0028] In operation, the assembled, two-chip device is engaged with
a switching element that contains multiple channels, such as the
rotor shown in FIG. 1B. A sample is loaded into inlet port 111,
which is the inlet to the deglycosylation column 110. The reaction
mixture after deglycosylation flows, sequentially, to inlet/outlet
port 141, inlet/outlet port 141' on chip 210 which is connected to
port 141, a channel in the rotor that connects port 141' with port
142, port 142, and trapping column 120. After trapping, the rotor
is switched to allow fluid communication between trapping column
120 and the separation column 130 (through a channel in the rotor
that connects port 144 and port 145), and the glycans are eluted
and separated as described above.
[0029] FIGS. 3 and 4 show three-dimensional views of how some
embodiments of two-layer devices work in conjunction with a
switching element. As illustrated in FIG. 3, the sample is injected
into inlet port 111 to enter the deglycosylation column, which is
in the shape of a loop in the embodiment in this figure. Only the
beginning part and the ending part of the loop are shown. This
particular embodiment differs from the one shown in FIG. 2 in that
port 141 is below inlet port 111, which is a variation that serves
the same function. The output from the deglycosylation column is
led into the trapping column through channel 151, and glycan
enrichment occurs in the trapping column, which is in a lower layer
compared to the deglycosylation column. Proteins without a
carbohydrate moiety flow through the trapping column and exit
through channel 155. In FIG. 4, the trapping column is connected
with channels 151 and 153 (by switching a switching element not
completely shown in the figure), an elution solution is applied to
the trapping column through channel 151, and glycans are eluted
from the trapping column and enters the separation column through
channel 153.
[0030] In some embodiments, the device further comprises a cleaning
column that is capable of preferentially binding proteins but not
carbohydrates. For example, the C18, C8, or other reverse phase
material retains protein while passing free glycans. For proteins
such as antibodies, protein A can be used to bind the antibody but
not the glycans. The proteins bound to the cleaning column can be
discarded or analyzed to understand the protein profile and content
in the sample. These embodiments have inlet/outlet ports that, when
coupled with a switching element, allow for connection between the
deglycosylation column and the cleaning column, and between the
cleaning column and the trapping column, either at the same time or
not. It is also contemplated that in some embodiments, the
deglycosylation column can be connected with the trapping column,
and the trapping column with the cleaning column, either at the
same time or not. Thus, in some embodiments, all of these three
columns can be connected at the same time in the order of
deglycosylation-cleaning-trapping, or
deglycosylation-trapping-cleaning.
[0031] FIGS. 5 and 6 illustrate a three-dimensional view of a
device that comprises a cleaning column in a three-layer design.
The first layer includes a deglycosylation column 110, a protein
cleaning column 115 is in the middle layer, and the bottom layer
contains a trapping column 120 and a separation column 130. The
figures also show three channels from a switching element.
[0032] Example 1 describes experiments in which the embodiment
depicted in FIGS. 5 and 6 was used to analyze glycan compositions
in an antibody sample. The results demonstrate that the device of
the present invention enables a fast analysis, and it is possible
to preserve the original chemical structures of the glycans in the
fast analysis. In contrast, a traditional in-solution analysis
performed in parallel indicates that chemical structures of the
glycans changed during the prolonged incubation of the traditional
analysis. Unexpectedly, isoforms of the glycans, now in their
original structures (amino glycan forms), can be resolved much more
readily in the separation column, whereas it had been necessary to
employ a long and more time-consuming and thus expensive separation
process to separate the isoforms, a problem that had been
troublesome in the field.
[0033] The speed of the present methods depends on complexity of
the samples, as longer separation is required when more glycans
need to be separated. In addition, if finer separation is desired,
such as separation of isomers, a longer separation column and thus
longer separation time is needed as well. By the present methods,
the process of deglycosylation, cleaning, trapping and separation
can generally be achieved in one minute to an hour, such as in 1,
2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes.
In some embodiments, the process is performed under conditions that
allow a substantial amount of the glycans to remain in amino glycan
forms. This substantial amount may be, for example, at least 95,
90, 80, 70, 60, 50, 40, or 30% of the glycans in the sample.
[0034] The results also indicate that a small amount of sample (100
ng glycoproteins) yielded strong signals, suggesting that the
amount of the sample can be reduced. Thus, in some embodiments, the
present methods can be used to analyze a sample containing up to
10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 ng of glycoproteins.
[0035] Thus, some embodiments of the present invention provide
devices, usually in the form of chips, that comprise:
[0036] a deglycosylation column comprising a solid support and an
enzyme immobilized to the solid support, wherein the enzyme is
capable of cleaving carbohydrates from glycoproteins;
[0037] a trapping column capable of binding carbohydrate;
[0038] a separation column capable of separating carbohydrates;
and
[0039] a plurality of inlet/outlet ports;
[0040] wherein said ports are configured so that when said device
is aligned with a switching element that comprises a channel or a
plurality of channels, the combination of said ports, columns and
channel(s) forms a valve system that can be switched between at
least a first state and a second state, the first state allowing
fluid communication between the deglycosylation column and the
trapping column, and the second state allowing fluid communication
between the trapping column and the separation column.
[0041] The device can optionally further comprise a cleaning column
that is capable of binding proteins. The cleaning column may be
used to bind and discard proteins, which are undesirable in glycan
analyses. Alternatively, the cleaning column can be used to collect
proteins for a protein analysis in addition to the glycan
analysis.
[0042] In some other embodiments, the device may comprise a
deglycosylation column, a cleaning column, and a separation column,
without the trapping column. These embodiments fall into two
groups. One group can be used for protein analyses, in which case
the separation column is one for separation of proteins rather than
carbohydrates. A switching element capable of connecting the
cleaning column with the separation column is to be used in
conjunction with this group. The other group can be used for a
shortened glycan analysis process, in which glycan enrichment is
omitted. Embodiments in this group comprise a separation column for
separating carbohydrates.
[0043] Microfluidic devices are typically fabricated by generating
channels and chambers in a material that forms the device or layers
of the device. Suitable materials for forming the device, or layers
of the device, are selected with regard to physical and chemical
characteristics that are desirable for proper functioning of the
microfluidic device. For example, a device is fabricated from a
material that enables formation of high definition features, i.e.,
microchannels, microchambers and the like, that are of micron or
submicron dimensions. Thus, the material must be capable of
microfabrication using, e.g., dry etching, wet etching, laser
etching, laser ablation, molding, embossing, or the like, so as to
have desired miniaturized surface features. Microstructures can
also be formed on the surface of a material by adding material
thereto, for example, polymer channels can be formed on the surface
of a glass substrate using photo-imageable polyimide. Also, all
device materials used should be chemically inert and physically
stable with respect to any substance with which they comes into
contact when used to introduce a fluid sample (e.g., with respect
to pH, electric fields, etc.). Suitable materials for forming the
present devices include, but are not limited to, polymeric
materials, ceramics (including aluminum oxide and the like), glass,
metals, composites, and laminates thereof.
[0044] Polymeric materials are typically organic polymers that are
homopolymers or copolymers, naturally occurring or synthetic,
crosslinked or uncrosslinked. Specific polymers of interest
include, but are not limited to, polyimides, polycarbonates,
polyesters, polyamides, polyethers, polyurethanes,
polyfluorocarbons, polystyrenes,
poly(acrylonitrile-butadiene-styrene)(ABS), acrylate and acrylic
acid polymers such as polymethyl methacrylate, and other
substituted and unsubstituted polyolefins, and copolymers thereof.
Polyimide is of particular interest and has proven to be a highly
desirable substrate material in a number of contexts.
Polyetheretherketones (PEEK) also exhibit desirable biofouling
resistant properties.
[0045] The devices of the invention may also be fabricated from a
"composite," i.e., a composition comprised of unlike materials. The
composite may be a block composite, e.g., an A-B-A block composite,
an A-B-C block composite, or the like. Alternatively, the composite
may be a heterogeneous combination of materials, i.e., in which the
materials are distinct from separate phases, or a homogeneous
combination of unlike materials. As used herein, the term
"composite" is used to include a "laminate" composite. A "laminate"
refers to a composite material formed from several different bonded
layers of identical or different materials. Other preferred
composite materials include polymer laminates, polymer-metal
laminates, e.g., polymer coated with copper, a ceramic-in-metal or
a polymer-in-metal composite. One preferred composite material is a
polyimide laminate formed from a first layer of polyimide such as
Kapton.RTM., that has been co-extruded with a second, thin layer of
a thermal adhesive form of polyimide known as KJ.RTM., both
available from DuPont (Wilmington, Del.).
[0046] The present devices can be fabricated using any convenient
method, including, but not limited to, micromolding and casting
techniques, embossing methods, surface micro-machining and
bulk-micromachining. The latter technique involves formation of
microstructures by etching directly into a bulk material, typically
using wet chemical etching or reactive ion etching ("RIE"). Surface
micro-machining involves fabrication from films deposited on the
surface of a substrate. An exemplary surface micro-machining
process is known as "LIGA." See, for example, Becker et al. (1986),
Ehrfeld et al. (1988), and Guckel et al. (1991). LIGA involves
deposition of a relatively thick layer of an X-ray resist on a
substrate followed by exposure to high-energy X-ray radiation
through an X-ray mask, and removal of the irradiated resist
portions using a chemical developer.
[0047] Another technique for preparing the present devices is laser
ablation. In laser ablation, short pulses of intense ultraviolet
light are absorbed in a thin surface layer of material. Preferred
pulse energies are greater than about 100 millijoules per square
centimeter and pulse durations are shorter than about 1
microsecond. Under these conditions, the intense ultraviolet light
photo-dissociates the chemical bonds in the substrate surface. The
absorbed ultraviolet energy is concentrated in such a small volume
of material that it rapidly heats the dissociated fragments and
ejects them away from the substrate surface. Because these
processes occur so quickly, there is no time for heat to propagate
to the surrounding material. As a result, the surrounding region is
not melted or otherwise damaged, and the perimeter of ablated
features can replicate the shape of the incident optical beam with
precision on the scale of about one micron or less. Laser ablation
will typically involve use of a high-energy photon laser such as an
excimer laser of the F.sub.2, ArF, KrCl, KrF, or XeCl type.
However, other ultraviolet light sources with substantially the
same optical wavelengths and energy densities may be used as well.
Laser ablation techniques are described, for example, by Znotins et
al. (1987), and in U.S. Pat. Nos. 5,291,226 and 5,305,015 to
Schantz et al.
[0048] Another aspect of this invention provides systems that
comprise a device of the present invention. The system may further
comprise a switching element for switching a valve system to allow
selective connections between the various columns of the device.
The switching element may be, for example, a rotor comprising a
plurality of channels that can be aligned with the inlets/outlet
ports of the chip when the chip is engaged with the rotor.
Furthermore, the rotor can be switched between at least two
different states, each of which allows for connection of different
ports in the chip. The switching element may also be a sliding
element that can be slid between at least two different positions
to connect different ports, such as those described in U.S. Pat.
No. 6,702,256. Other switching mechanisms can also be used. The
system may optionally comprise an interface for transmitting the
output of the device (separated glycans) to the inlet of a mass
spectrometer or other analysis tools. Alternatively, the output of
the device may directly enter the ionization area of a mass
spectrometer. The system may further comprise a mass spectrometer
or other analysis tools.
[0049] Other embodiments also provide kits that comprise a device
of the present invention. The kit can further comprise any one or
any combination of the following: sample solution, elution solution
for any of the columns, reconstitution reagents for any of the
columns, a standard sample (such as an antibody standard, glycan
standard, etc.), instructions for use, and a container for the
components.
[0050] The following examples are offered to illustrate various
embodiments of this invention and are not to be construed in any
way as limiting the scope of the present invention. While certain
embodiments of this invention are particularly shown and described,
various changes in form and details may be made therein without
departing from the spirit and scope of the invention.
EXAMPLES
[0051] In the examples below, the following abbreviations have the
following meanings. Abbreviations not defined have their generally
accepted meanings.
[0052] .degree. C.=degree Celsius
[0053] hr=hour
[0054] min=minute
[0055] sec=second
[0056] mM=millimolar
[0057] .mu.M=micromolar
[0058] nM=nanomolar
[0059] .mu.m=micrometer
[0060] ml=milliliter
[0061] .mu.l=microliter
[0062] nl=nanoliter
[0063] mg=milligram
[0064] .mu.g=microgram
[0065] HPLC=high performance liquid chromatography
[0066] LC=liquid chromatography
[0067] MS=mass spectrometry
[0068] QTOF=quadrupole-time of flight
Example 1
Glycan Analysis with On-Chip Enzymatic Reactions
Chip Fabrication
[0069] PNGase F enzyme was chemically attached to epoxide-modified
5 .mu.m silica beads in phosphate buffer (pH 8) over 22 hr. The
beads were then packed into a chamber located in a first polyimide
chip. Reverse-phase 5-.mu.m C8 beads were packed into a second
chamber on a second polyimide chip for protein cleaning. The third
chip layer contained a sample enrichment column (40 nl), a
separation column (75 mm) filled with graphitized carbon for HPLC
analysis, and an integrated MS tip for input into a mass
spectrometer. All three chips were stacked, aligned and inserted
into a chip frame holder.
Methods
[0070] The antibody samples were diluted in phosphate buffer to a
concentration of 6 pmol/.mu.l in phosphate buffer (pH 7.5). The
HPLC system operated using typical mobile phases of 0.1% formic
acid in MS-grade water (Solvent A) and 0.1% formic acid in
acetonitrile (Solvent B). The autosampler was set to inject 0.1
.mu.l of antibody. The capillary pump was set to pass the sample
through the deglycosylation column and the C8 column at a flow rate
of 1 .mu.l/min. This flow rate corresponds to a deglycosylation
time of 8 seconds, and resulted in 100% deglycosylation. The
solvent used for loading and deglycosylation was 50 mM sodium
phosphate. The gradient for fast deglycosylation and glycan
analysis was 2-32% Solvent B in 1.5 min, 32-75% Solvent B in 30 sec
and held at 75% Solvent B for another 30 sec, followed by 75-2%
Solvent B over the last 30 sec. with a nano pump flow of 300
nl/min. A longer gradient was used to separate isomers.
[0071] The separated glycans were analyzed using a QTOF tandem mass
spectrometer with an electrospray ionization source. The mass
spectra were recorded in positive ion mode. Voltage of the cap was
set to 1750V, with a drying gas of 325.degree. C. at 8 L/min. The
fragmentation voltage was 120V. The skimmer voltage was 65V. Data
was acquired in 2 GHz mode.
Results
[0072] Antibody samples were deglycosylated and the resulting
glycans were separated by HPLC and analyzed by mass spectrometry,
using the chip and method as described above. The expected glycans
include any of the following:
TABLE-US-00001 TABLE 1 Candidate glycans Main ion species [m/z + 2]
Name Structure Glycan Mass + 2 G0 ##STR00001## 1462.54 732.27 G1'
##STR00002## 1624.59 813.29 G1'' ##STR00003## G2 ##STR00004##
1786.65 894.32 Mann-5 ##STR00005## 1234.43 618.21 Open circle:
galactose Square: GlcNAc Closed circle: Mannose Triangle:
Fucose
[0073] FIG. 7 (left-hand side) shows a typical result from the
traditional, in-solution analysis in which the sample was cleaved
by PNGase F in solution overnight in a protocol that lasted 24
hours in total. Of the major peaks, 303 and 305 correspond to two
stereoisomers (.alpha.- and .beta.-anomers) of G0, and 304 and 306
correspond to the two isomers of G1, G1' and G1'' (see Table 1
above). All these glycan species eluted around 3.4-3.7 minutes. In
addition, there are also minor peaks 301 and 302 eluting around 3.2
minutes. However, the results of the on-chip analysis using the
same sample were different. As shown in FIG. 7, right-hand side,
the sample yielded a prominent peak (310) and two smaller peaks
(311 and 312), all around 3.2 minutes. Four minor peaks 313-316
appeared between 3.4 and 3.7 minutes, where the major peaks in the
in-solution analysis were. In fact, the positions of 313-316 were
in good agreement with peaks 303-306 in the in-solution analysis,
just like peaks 310 and 311 eluted at about the same times as peaks
301 and 302.
[0074] The discrepancy between the in-solution and on-chip results
can be explained in view of the mechanism of action of PNGase F and
the efficient, on-line nature of the chip. According to Rasmussen
et al., 1992 (see FIG. 8), PNGase F acts by first cleaving the C--N
bond of the glycosylated asparagine side chain, and the asparagine
residue is converted to aspartic acid. The cleaved glycan is an
amino glycan initially, but the amino group is slowly hydrolyzed to
a hydroxyl group with the release of ammonia. Taking into account
the masses of each peak in FIG. 7, we postulated that peak 310
corresponds to amino glycan of G0, which is hydrolyzed to hydroxyl
glycans (peaks 313 and 315) upon a long incubation. To test this
hypothesis, a time-course experiment was conducted in which the
sample was allowed to stay in the deglycosylation column in our
chip for 0, 15, 30, 60, 120 and 240 minutes, respectively. Indeed,
peak 310 decreased and peaks 313 and 315 increased in intensity
with each prolonged incubation time. Similarly, the peaks for G1 in
the in-solution analysis (304 and 306) also decreased over time,
and 311 increased. Thus, based on the time-dependent changes and
masses of the peaks, we conclude (see FIG. 9) that peaks 310, 311
and 312 correspond to amino glycans, and peaks 313-316 correspond
to hydroxyl glycans. There are two peaks (313 and 315, and 314 and
316) for each hydroxyl glycan, because hydroxyl glycans isomerize
easily between the .alpha.-anomer and the .beta.-anomer.
[0075] The on-line nature of the chip also contributes to
efficiency of the reaction. When a sample enters the
deglycosylation column and moves along, each "front" of the sample
comes in contact with an excess amount of the enzyme and is cleaved
instantly. On the other hand, the in-solution incubation mixture
contains a lower enzyme to substrate ratio for each substrate, and
the reaction tends to leave a population of various incompletely
digested products. Furthermore, in the on-line process, the cleaved
glycans continue to move along, bind to the trapping column, and
become separated in the separation column. Thus, the glycans are
bound by other molecules most of the time and have less chance to
isomerize or undergo other chemical reactions.
[0076] The ability of the present invention to catch the amino
glycans makes it possible to separate the two isoforms of G1
efficiently. G1 exists as two isomers (G1' and G1'' in Table 1). It
was believed that G1' and G1'' could only be separated using a very
long gradient. Therefore, it has been a problem to accurately
analyze glycan compositions without spending the resources and time
for a long separation step. We discovered, however, that the amino
forms can be readily separated. The following short gradient was
used to run the separation column in the chip described in this
example, and G1' and G1'' were clearly resolved (FIG. 10) in a
matter of minutes:
TABLE-US-00002 Time (min) % Solvent B 0 2 10 22 12 80 15 80 16
2
[0077] Thus, these results demonstrate that the chips described in
this disclosure not only significantly shorten the time required
for glycan analysis, but the data obtained from the chips reflect
the structures of the glycans more faithfully. In addition, only a
small amount of initial sample is required for the chip to achieve
the same results. The sample in this example contained only 100 ng
of glycoproteins, and the signals in mass spectra were very high.
Therefore, a lower amount of sample can be used.
Exemplary Embodiments
[0078] Embodiments of the present invention include, without being
limited to, the following: [0079] A. A microfluidic device for
removing carbohydrate from a glycoprotein, comprising:
[0080] a deglycosylation column comprising a solid support and an
enzyme immobilized to the solid support, wherein the enzyme is
capable of cleaving carbohydrates from glycoproteins;
[0081] a trapping column that is capable of binding
carbohydrates;
[0082] a separation column capable of separating carbohydrates;
and
[0083] a plurality of inlet/outlet ports;
wherein said ports are configured so that when said device is
coupled with a switching element that comprises at least one
channel, the combination of said ports, columns and at least one
channel forms a valve system that can be switched between at least
a first state and a second state, the first state allowing fluid
communication between the deglycosylation column and the trapping
column, and the second state allowing fluid communication between
the trapping column and the separation column. [0084] B. The device
of embodiment A, further comprising a cleaning column capable of
binding proteins, wherein the cleaning column is configured to be
connectable to the deglycosylation column and/or the trapping
column by the valve system. [0085] C. The device of embodiment A or
B, wherein the enzyme is N-glycosidase F. [0086] D. The device of
any one of embodiments A-C, wherein the solid support in the
deglycosylation column comprises beads or a monolithic medium.
[0087] E. The device of any one of embodiments A-D, wherein the
separation column is a liquid chromatography column. [0088] F. The
device of any one of embodiments A-D, wherein the separation column
is a capillary electrophoresis apparatus. [0089] G. The device of
any one of embodiments A-F that comprises two layers, wherein the
deglycosylation column is in one layer, and the trapping column and
separation column are in the other layer. [0090] H. The device of
any one of embodiments B-F that comprises three layers, wherein the
deglycosylation column is in a first layer, the cleaning column is
in a second layer, and the trapping column and separation column
are in a third layer. [0091] I. A system for analyzing a sample,
comprising the device of any one of embodiments A-H, the switching
element, and a mass spectrometer. [0092] J. The system of
embodiment I, wherein the mass spectrometer comprises an
electrospray ion source. [0093] K. A method for analyzing the
carbohydrate moieties of glycoproteins, comprising:
[0094] applying a sample that may comprise glycoproteins to the
device of embodiment A;
[0095] digesting the glycoproteins in the deglycosylation column to
result in cleaved carbohydrates;
[0096] binding the cleaved carbohydrates to the trapping
column;
[0097] eluting the cleaved carbohydrates from the trapping column;
and
[0098] separating the cleaved carbohydrates with the separation
column. [0099] L. The method of embodiment K, further comprising
removing proteins after the digesting with a cleaning column
capable of binding proteins. [0100] M. The method of embodiment K
or L, wherein the cleaved carbohydrates are separated by liquid
chromatography. [0101] N. The method of embodiment K or L, wherein
the cleaved carbohydrates are separated by capillary
electrophoresis. [0102] O. The method of any one of embodiments
K-N, further comprising analyzing the cleaved carbohydrates using
mass spectrometry. [0103] P. The method of any one of embodiments
K-O, wherein the sample contains up to 50 ng of glycoproteins.
[0104] Q. The method of any one of embodiments K-P, wherein the
method is completed within 10 minutes. [0105] R. The method of any
one of embodiments K-Q that is performed under conditions that
allow at least some of the cleaved carbohydrates to remain in amino
glycan forms. Preferably, a substantial amount of the cleaved
carbohydrates remain in amino glycan forms. [0106] S. The method of
any one of embodiments K-R, wherein the glycoproteins are digested
with N-glycosidase F. [0107] T. A kit for glycan analysis,
comprising the device of any one of embodiments A-H and at least
one reagent for sample dilution or column elution.
REFERENCES
[0108] Palm and Novotny (2005), "A monolithic PNGase F enzyme
microreactor enabling glycan mass mapping of glycoproteins by mass
spectrometry," Rapid Comm. Mass Spectrometry 19: 1730-1738.
[0109] Rasmussen et al. (1992), "Identification and derivatization
of (oligosaccharyl)amines obtained by treatment of
asparagine-linked glycopeptides with N-GLYCANASE enzyme," J. Am.
Chem. Soc., 114(3): 1124-1126.
[0110] Becker et al. (1986), "Fabrication of Microstructures with
High Aspect Ratios and Great Structural Heights by Synchrotron
Radiation Lithography Galvanoforming, and Plastic Moulding (LIGA
Process)," Microelectronic Engineering 4(1):35-36.
[0111] Ehrfeld et al. (1988), "1988 LIGA Process: Sensor
Construction Techniques via X-Ray Lithography," Tech. Digest from
IEEE Solid-State Sensor and Actuator Workshop, Hilton Head,
S.C.
[0112] Guckel et al. (1991), J. Micromech. Microeng. 1:
135-138.
[0113] Znotins et al. (1987), Laser Focus Electro Optics, at pp.
54-70.
[0114] U.S. Pat. Nos. 6,702,256; 5,291,226 and 5,305,015.
[0115] U.S. Patent Publication No. 2006/0171855.
[0116] All of the publications, patents and patent applications
cited above or elsewhere in this application are herein
incorporated by reference in their entirety to the same extent as
if the disclosure of each individual publication, patent
application or patent was specifically and individually indicated
to be incorporated by reference in its entirety.
[0117] A number of embodiments of the invention have been
described. Nevertheless, it will be understood that various
modifications may be made without departing from the spirit and
scope of the invention.
* * * * *